In 2007 it was reported that over half a million people died from cancer and more than 1.4 million new cases were diagnosed in the United States. Thus, it is important to diagnose, assess prognosis for, and monitor treatment of cancer. Improved, noninvasive diagnostic compositions and methods using cancer-specific markers that provide strong discrimination between biological samples from cancer patients and normal individuals are especially needed in the art.
The nucleotide salvage pathway is a pathway in which nucleotides (purines and pyrimidines) are synthesized from intermediates in the degradative pathway for nucleotides. Salvage pathway enzymes are used to recover bases and nucleosides that are formed during degradation of RNA and DNA. This is important because some tissues cannot undergo de novo synthesis. There are two major pathways that lead to the production of nucleotides: the de novo pathway and the salvage pathway. The de novo synthesis of DNA begins with metabolic precursors such as amino acids, ribose 5-phosphate, and others known to the art. Most organisms also synthesize nucleotides through a salvage pathway that utilizes the degraded nucleotides from DNA, recycling nucleic acids rather than destroying them, providing an energy efficient way to produce nucleotides. This is accomplished through the enzymatic activities of adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), for purines, and thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) for pyrimidines, producing oligonucleotides which are further digested by phosphodiesterases to yield free nucleosides.
The de novo pathway to deoxythymidine triphosphate (dTTP) synthesis first requires the use of deoxyuridine monophosphate (dUMP) from the metabolism of either uridine diphosphate (UDP) or cytidine diphosphate (CDP). The dUMP is converted to deoxythymidine monophosphate (dTMP) by the action of thymidylate synthase. The methyl group of thymine is donated by N5,N10-methylene tetrahydrofolate (THF), similar to the donation of methyl groups during the biosynthesis of the purines. The unique property of the action of thymidylate synthase is that the THF is converted to dihydrofolate (DHF), the only such reaction yielding DHF from THF. In order for the thymidylate synthase reaction to continue, THF must be regenerated from DHF. This is accomplished through the action of dihydrofolate reductase (DHFR). THF is then converted to N5,N10-THF via the action of serine hydroxymethyl transferase.
Expression of genes related to DNA replication is maximal during early S phase. TK1, APRT, HGPRT and dCK are cellular enzymes involved in a salvage pathway of DNA synthesis. In normal growing cells these salvage pathway enzymes' mRNA levels rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1. They are activated in the G1/S phase of the cell cycle. DNA synthesis is primarily dependent upon salvage mechanisms to supply deoxypyrimidine nucleotides. Antisense inhibition of the de novo pathway of pyrimidine synthesis results in a compensatory stimulation of the less energy-consuming salvage pathways. The salvaged bases and nucleosides can then be converted back into nucleotides.
Thymidine kinase 1 (TK1) is an S-phase-regulated specific protein which is found both in normal individuals and in cancer patients. TK1 catalyses the reaction of thymidine to deoxythymidine monophosphate (dTMP) using adenosine triphosphate (ATP), and dCK works similarly with cytidine as its substrate. APRT and HGPRT both use phosphoribosyl pyrophosphate to phosphorylate adenine and guanine to adenosine monophosphate (AMP) and guanosine monophosphate (GMP) respectively.
Substantial research has been performed on TK1, but comparatively little research has been done on the other nucleotide salvage pathway enzymes as related to cancer. Mammalian cells express two different isoenzymes, TK1 and TK2, originally called fetal and adult TK, respectively. TK1 was originally called fetal TK since it was predominantly found in the fetus. The fetal isoenzyme, TK1, is associated with cell division, and its levels increase as the cell is dividing.
U.S. Pat. No. 5,698,409 (incorporated herein by reference) describes a mammalian TK1 purified from Raji cells and a TK1-specific monoclonal antibody. The monoclonal antibody binds to TK1 and inhibits TK1 activity. The TK1 monoclonal antibody was used for cancer diagnosis. U.S. Pat. Nos. 7,311,906 and 7,837,998, incorporated herein by reference, refer to TK1-specific monoclonal antibodies produced by hybridomas on deposit with American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Va., under Accession Nos. ATCC HB 11432, HB 11433, HB 11434 and PTA-670.
Other references of interest are listed in the References section below and incorporated herein by reference for purposes of enablement and written description.